This invention relates to a non-naturally occurring immunogenic polypeptide derived from a merozoite antigen of Plasmodium species, and to methods of production thereof. The invention also relates to the use of this immunogenic polypeptide in a subunit vaccine against malaria.
Apical membrane antigen 1 (AMA-1) of Plasmodium species (previously also referred to as RMA-1) is a merozoite protein that is considered a leading candidate for inclusion in a malaria vaccine (see International Patent Publication No. WO 89/07645). AMA-1 is found associated with the merozoite apical organelles but subsequently it relocates to the merozoite surface prior to merozoite invasion of the erythrocyte. Early studies with PK66, the homologue of AMA-1 expressed in the simian parasite Plasmodium knowlesi, showed that merozoite invasion could be inhibited with a monoclonal antibody to AMA-11,2 and that immunization with the purified parasite antigen provided partial protection of rhesus monkeys against infection with P. knowles3.
The AMA-1 gene sequence has been determined for a number of different isolates of P. falciparum4,5 and for several other Plasmodium species6-8. The deduced amino acid sequence is typical of a type I integral membrane protein with an NH2-terminal signal sequence and a presumed transmembrane domain towards the COOH-terminus. A comparison of the various AMA-1 sequences shows this to be a relatively conserved protein which contrasts with MSA-1 and MSA-2, two other well characterised merozoite surface antigens. In the large NH2-terminal, presumably ectodomain, there are 16 cysteine residues that are conserved in all AMA-1 sequences. This indication that folding of the ectodomain is stabilised by intramolecular disulphide bonds is supported by the observation that the mobility of AMA-1 on SDS-PAGE varies depending on whether or not the sample buffer contains a reducing agent.
Because AMA-1 is relatively conserved in the genus it has been possible to identify clones corresponding to the homologue in other species of Plasmodium. Thus, AMA-1 of the simian malaria, P. fragile7 and the murine malaria P. chabaudi adami6 are available for studies of vaccine efficacy using appropriate host-parasite combinations. Because of the presumed disulphide bonded structure, the AMA-1s of both P. fragile and P. chabaudi were expressed in insect cells using recombinant baculovirus to produce antigen for use in preclinical vaccine trials. The P. fragile AMA-1 expressed in this way was isolated by lentil lectin chromatography and then anion- and cation-exchange chromatography. Saimiri monkeys immunized with the semi-purified molecule were partially protected against challenge with P. fragile. In this trial the degree of protection among immunized monkeys was positively correlated with the titre of antibodies induced by immunization. All surviving monkeys were drug-treated to eliminate persisting parasitaemias and re-challenged with P. falciparum. Three of four control monkeys developed transient, low parasitaemias whereas no P. falciparum parasitaemias were detected in the five immunized animals that were re-challenged. Thus, immunization with P. fragile AMA-1 provides protection against the homologous parasite and also may provide some cross-immunity against P. falciparum over and above that provided by a prior infection with P. fragile. 
Subsequently, the protective efficacy of AMA-1 was studied in mice using the P. chabaudi homologue of AMA-1. Initially, the full-length P. chabaudi AMA-1 was expressed in insect cells using recombinant baculovirus and a vaccine trial was carried out using a lysate of insect cells infected with the recombinant baculovirus (In preliminary experiments cell lysates induced antibody responses that approached those induced by the semi-purified P. fragile antigen). C3H/He mice immunized with insect cell lysates containing the P. chabaudi AMA-1 were protected against challenge with P. chabaudi. None of the immunized mice died and peak parasitaemias were very much lower than those in mice that were immunized with lysates of insect cells infected with non-recombinant baculovirus.
Although it may be possible to base a vaccine against P. falciparum infections of humans on baculovirus-expressed full-length P. falciparum AMA-1, there are several disadvantages to using this approach for a human vaccine. First, it is more expensive to use a eukaryotic expression system than it is to use E. coli as the host cell. Second, the antigen is glycosylated in insect cells and this introduces microheterogeneity into the protein which could make it difficult to manufacture reproducible batches of purified material. Third, AMA-1 is a type I integral membrane protein and therefore the full-length molecule would be difficult to work with in aqueous buffers without added detergent. In view of the above disadvantages, the present inventors have examined the possibility of producing AMA-1 in E. coli in a form soluble in aqueous buffers and capable of inducing a protective immune response.
According to the present invention there is provided a non-naturally occurring immunogenic polypeptide comprising an amino acid sequence corresponding to a non-full length fragment of the apical membrane antigen 1 (AMA-1) of Plasmodium species, said polypeptide not including an amino acid sequence corresponding to the transmembrane domain of AMA-1 and being stabilised by folding.
Folding of the polypeptide is preferably achieved by generation of intramolecular disulphide bonds which stabilise the polypeptide in a conformation required for inducing a protective immune response.
The amino acid sequence of the immunogenic polypeptide corresponding to a non-full length fragment of AMA-1, may be a fragment corresponding to the mature ectodomain of the antigen not including the transmembrane domain thereof, or a portion thereof.
Preferably, the AMA-1 fragment is a fragment of P. falciparum AMA-1, however the present invention also extends to AMA-1 fragments of other Plasmodium species, including P. vivax, P. fragile and P. chabaudi adami 
Preferably also, the AMA-1 fragment is a fragment which contains at least two of the conserved cysteine residues of the AMA-1 molecule. It will be appreciated, however, that the fragment may contain from two to all of the sixteen conserved cysteine residues of AMA-1.
The immunogenic polypeptide in accordance with this invention is preferably produced by recombinant DNA technology.
Accordingly, in another aspect the present invention provides a recombinant DNA molecule comprising a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence corresponding to a non-full length fragment of the apical membrane antigen 1 (AMA-1) of Plasmodium species, said polypeptide not including an amino acid sequence corresponding to the transmembrane domain of AMA-1.
The recombinant DNA molecule may also comprise an expression control sequence operatively linked to the nucleotide sequence as described above.
The present invention also extends to a recombinant DNA cloning vector containing a recombinant DNA molecule as broadly described above, as well as to a host cell such as E. coli containing such a recombinant DNA molecule or recombinant DNA cloning vector. Such a host cell, of course, provides means for the production of the immunogenic polypeptide of the present invention using techniques which are well known to persons skilled in this art.
As an alternative to the prokaryotic host cell, the recombinant DNA molecule of this invention may be cloned using a eukaryotic host cell such as yeast, with folding and disulphide bond formation being achieved by passage of the expressed polypeptide through the secretory pathway of the cells.
It has been found that a stabilised conformation is required to enable the AMA-1 fragment to induce a protective response, and accordingly where the immunogenic fragment is produced as a recombinant product, particularly in a prokaryotic expression system, refolding of the product with disulphide bond formation is required to produce an efficacious immunogenic polypeptide.
In work leading to the present invention, the present inventors have discovered a fragment of AMA-1 corresponding to the mature ectodomain which, when expressed in E. coli, purified by Ni-chelate chromatography, refolded by dilution in the presence of reduced and oxidized glutathione, concentrated by anion-exchange chromatography, and injected formulated with an appropriate adjuvant, induces an immune response which protects against malaria.
Accordingly, the present invention extends to a vaccine composition, or composition for inducing an immune response in a host, comprising an immunogenic polypeptide as broadly described above, and a pharmaceutically acceptable carrier or diluent. Such a vaccine composition preferably also comprises an adjuvant.
The formulation of such vaccine compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The present invention also provides a method for actively immunizing a host against Plasmodium infection, which comprises administering to the host an effective amount of a vaccine composition as described above.
The active component is administered in prophylactically effective amounts. A prophylactically effective amount means that amount necessary at least partly to attain the desired effect, that is to induce an immune response against Plasmodium infection. Such amounts will depend, of course, on individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
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Studies with P. chabaudi AMA-1 have provided evidence that a fragment of AMA-1 corresponding to the mature ectodomain, when expressed in E. coli, purified by Ni-chelate chromatography, and refolded by dilution in the presence of reduced and oxidized glutathione is an antigen of particular interest as a potential subunit vaccine against human malaria. The results indicate that the NH2-terminal domain (presumed ectodomain) of AMA-1 alone is sufficient to induce a protective immune response. Thus, there is no requirement to express the full-length polypeptide which, being a type I integral membrane protein, has solubility characteristics that make it difficult to purify using aqueous buffers and conventional chromatographic procedures. The results also indicate that the ectodomain of AMA-1 has a conformation stabilised by intramolecular disulphide bonds and that this conformation (or a related conformation stabilised by disulphide bonds) is required to induce a protective immune response. Because of the reducing environment in the cytosol of bacteria, recombinant proteins expressed in E. coli or other prokaryotic host cells usually do not fold with the correct disulphide-bonding pattern. Because of this, it is usual to utilise eukaryotic expression systems to generate appropriately folded proteins with multiple intramolecular disulphide bonds. Although the disulphide bonding pattern in AMA-1 has not been established, it is clear that the refolding procedure used in these studies results in the formation of intramolecular disulphide bonds that are critical for generating epitopes recognized by antibodies induced by malaria infections of mice. As a reduced and alkylated protein was incapable of inducing a protective immune response, it is also clear that intramolecular disulphide bonds generated in the E. coli-expressed recombinant AMA-1 ectodomain are critical for creating epitopes that induce protective immune responses.